The invention relates generally to methods for determining activity and suitable dosage levels for antimicrobial and/or antiviral drugs to restrict selection of resistant mutants.
For any given microbial or viral pathogen (e.g., bacterial, fungal, or viral pathogen), there typically exists a characteristic concentration of a specified antimicrobial or antiviral drug (hereafter xe2x80x9cdrugxe2x80x9d), or combination of drugs, at which recovery of microbial colonies or viral plaques from drug-containing cultures sharply decreases. This concentration is referred to as the xe2x80x9cminimum inhibitory concentrationxe2x80x9d (MIC), and is conventionally defined with reference to a specific percent inhibition of pathogen growth. Thus, for example, the concentration of drug at which 99% of pathogen growth is inhibited is labelled MIC99. In spite of the sharp decrease in pathogen growth at the MIC, a small but finite fraction are often able to grow in the presence of the drug. These pathogens are termed xe2x80x9cdrug-resistant.xe2x80x9d Drug resistant mutants arise spontaneously within pathogen populations. When a pathogen population is treated with a drug for an extended period of time (e.g., one or more days), resistant mutants proliferate while drug-sensitive, wild-type cells do not. Eventually, the pathogen population becomes dominated by the resistant mutants. This process, which is called xe2x80x9cselectionxe2x80x9d, can occur both in vitro and in vivo. The selection process is responsible for the development of resistant mutants in, for example, infected human patients. The mutant pathogens can spread to other persons, resulting in an outbreak of disease unresponsive to the particular drug. It is then necessary to use an alternate drug to treat the disease. The alternate drug will similarly be useful only until mutant pathogens resistant to the alternate drug begin to proliferate and dominate the population.
In many cases, drug-resistant pathogens are resistant to only a single drug or class of drugs. In recent years, however, an alarmingly rapid increase has been observed in the number of pathogens that have become multi-drug resistant, meaning that they are resistant to two or more, and in some cases many, classes of drugs. It may be only a matter of time before some pathogens become resistant to all available drugs. Since it can take many years to develop a new drug, there is an urgent need to obtain reliable, quantitative methods for avoiding spread and further development of drug-resistant pathogens.
The problem of drug resistance is especially acute among immunocompromised patients. In these patients, blocking the growth of pathogens by using doses based on the MIC is not adequate to clear the infection; resistant mutants grow and can be transmitted from the infected person to others. As AIDS has spread through regions of the world where tuberculosis is widespread, for example, drug-resistant tuberculosis strains have emerged and rapidly spread when the drug-resistant bacteria have subsequently infected healthy (i.e., immunocompetent) persons. The diseases caused thereby have proven resistant to traditional treatments.
Drug dosing schedules are often based on a parameter called the area under the curve (AUC), where the curve represents a plot of drug concentration in human serum versus the time after delivery of the antibiotic or other drug into the human. One currently favored approach to dosing within the pharmaceutical industry involves the analysis of an empirical parameter called the AUIC, defined as the ratio of the AUC to minimum inhibitory concentration (MIC). No sound theoretical basis has yet been identified as to why a drug maintained at a particular multiple of the MIC should clear an infection. Moreover, the AUIC concept has not been demonstrated to have any relationship to drug resistance.
The invention is based on the discovery that, for many drugs (e.g., antiviral or antimicrobial drugs such as antifungal or antibiotic drugs, including bacteriocidal or bacteriostatic drugs) and many pathogen strains (e.g., viral, fungal, or bacterial pathogens), a concentration of drug can be identified at which drug-resistant mutant pathogen strains are not selected. This concentration is herein referred to as the xe2x80x9cmutant prevention concentrationxe2x80x9d (MPC). Maintaining serum concentrations of the drug above the MPC throughout a course of treatment should severely restrict selection of drug-resistant mutants. Additionally, it is discovered that drug-resistant mutant pathogens are selected exclusively within a drug concentration window, termed the xe2x80x9cmutant selection windowxe2x80x9d (MSW; FIG. 1). A quantitative expression of this window, which we call the xe2x80x9cwindow indexxe2x80x9d (WI), is defined as the ratio of the MPC to the MIC. The window index is characteristic of a given drug and a given pathogen.
In general, one embodiment of the invention features a method for determining the mutant prevention concentration (MPC) of a drug against a particular pathogen (e.g., a bacterial, fungal, or viral pathogen). The method includes the steps of obtaining a culture of said pathogen grown to high density (e.g., to stationary phase in the cases of bacteria and fungi); dividing at least some portion of the culture among a plurality (e.g., one, five, ten, one hundred, five hundred or more) of containers of a growth medium (e.g., agar plates) containing various concentrations of the drug; incubating the containers; counting the pathogen colonies (i.e., for bacteria or fungi), or plaques or foci (i.e., for viruses), if any, in the containers; plotting the number of counted colonies against drug concentration in each container; and, if necessary, extrapolating the plot to determine the minimum drug concentration corresponding to zero colonies. The minimum drug concentration corresponding to zero colonies is the MPC.
The invention also features a method for determining the mutant prevention concentration (MPC) of a drug against a particular pathogen (e.g., a bacterial, fungal, or viral pathogen). The method includes the steps of obtaining a culture of the pathogen, grown to high density (e.g., stationary phase), dividing at least some portion of the culture among a plurality (e.g., one, five, ten, one hundred, five hundred or more) of containers of a growth medium (e.g., agar plates, or a liquid culture broth) containing various concentrations of the drug; incubating the containers; and identifying the container having the lowest concentration of drug effective to prevent growth of the pathogen. This concentration is the MPC.
In any of the above methods: The culture can be concentrated or diluted, if necessary, prior to dividing among the containers. To refine measurement of the MPC, the method can be repeated with concentrations of drug more closely clustered around the MPC determined in the first iteration of the method.
The various concentrations of drug in the containers can differ from one another by a constant factor (e.g., ten-fold, five-fold, three-fold, or no more than about two-fold).
In the dividing step, between about 109 and about 1012 colony-forming units (e.g., between about 1010 and about 1012 colony-forming units) of bacteria can be divided among all of the containers that include a single concentration of the drug. The number of containers required for each concentration in order to apply this number of bacteria will be understood to depend on the size of each container, and can vary from one to 100 or more.
In another embodiment, the invention features a method for determining the window index (WI) (i.e., corresponding to the mutant selection window for a drug wherein resistant mutants of a specific pathogen are selected in the presence of the drug). The method includes the steps of determining the minimum inhibitory concentration (MIC) of the drug for the pathogen; determining the mutant prevention concentration (MPC) of the drug for the pathogen; and dividing MPC by MIC to obtain the WI. WI can optionally be added to or multiplied by various constants.
The invention also features an improved method for selecting a suitable drug for treating an infection caused by a specific pathogen (e.g., a bacterial, fungal, or viral pathogen), where the method includes determining an MIC. The improvement comprises determining the window index (WI). The method can also include the step of selecting at least one drug (e.g., 1, 2, 3 or more, 10 or more, or 100 or more drugs) having a toxicity level greater than its MPC for the specific pathogen. Further, the method can include selecting at least one drug (e.g., 1, 2, 3 or more, 10 or more, or 100 or more drugs) having a window index (WI) less than thirty (e.g., less than about 30, less than about 20, less than about 10, less than about 5, or less than about 2).
The invention also features an improved method for screening a plurality of compounds against a specific pathogen (e.g., a bacterial, fungal, or viral pathogen), where the method includes determining an MIC. The improvement comprises determining the window index (WI) The method can also include the step of selecting from the plurality at least one drug (e.g., 1, 2, 3 or more, 10 or more, or 100 or more drugs) having a toxicity level greater than its MPC for the specific pathogen. Further, the method can include selecting from the plurality at least one drug (e.g., 1, 2, 3 or more, 10 or more, or 100 or more drugs) having a window index (WI) less than thirty (e.g., less than about 30, less than about 20, less than about 10, less than about 5, or less than about 2).
In still another embodiment, the invention features a method of treating a patient infected with a pathogen (e.g., a bacterial, fungal, or viral pathogen). The method includes the steps of determining the MPC of a drug against the pathogen; and administering the drug to the patient at a dosage and frequency sufficient for the drug concentration in the patient""s serum to exceed the MPC. The dosage and frequency can be sufficient, for example, for Cmax to exceed the MPC, or sufficient to maintain serum concentration of the drug above the MPC in the patient for at least 5% (e.g., for at least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or about 100%) of the duration of the treatment.
The duration of treatment is determined by conventional methods and is generally unchanged by this invention. One object of the invention is to prevent mutants from arising and/or proliferating during a course of treatment; this will not generally necessitate altering the duration of treatment.
The drug can include, for example, at least one of a bacteriostatic drug, a bacteriocidal drug, an aminoglycoside, an anphenicol, an ansamycin, a xcex2-lactam, a lincosamide, a macrolide, a polypeptide antibiotic, a tetracycline, a cycloserine, a mupirocin, a tuberin, vancomycin, a 2,4-diaminopyrimidine, a nitrofuran, a quinolone, a fluoroquinolone, a sulfonamide, a sulfones, a fluoroquinolone, a macrolide, a tetracycline, an antifungal drug, an antiviral drug, or any derivative of any of these.
The patient can be, for example, a mammal (e.g., a human being) or a bird (e.g., a chicken). The drug can, for example, be administered in any form suitable for administration of drugs or medicaments (e.g., pills, tablets, chicken feed).
The invention also features a dosing regimen for a drug. The dosing regimen includes an administration level and frequency that maintains serum concentration in a treated subject below the toxicity level (e.g., cytotoxicity level CL) but above the MPC for at least 5% (e.g., for least 10%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or about 100%) of the time of a course of treatment.
As used herein, the term xe2x80x9cdrugxe2x80x9d is to be understood to include, for example, both antimicrobial drugs and antiviral drugs. xe2x80x9cAntimicrobial drugsxe2x80x9d include, for example, all antibiotics and other antibacterial agents (e.g., bacteriostatic and bacteriocidal drugs), as well as antifungal drugs.
The term xe2x80x9cpathogenxe2x80x9d herein refers to, for example, any bacterium, fungus, or virus that can infect a patient. A xe2x80x9cpatientxe2x80x9d can be, for example, a mammal (e.g., a primate such as a human; or a dog, a cat, a horse, a rabbit, a cow, a pig, or other mammal raised for human consumption or companionship), a fish (e.g., a salmon, a trout, a tuna, shellfish, a mollusk, or other fish raised for human consumption or companionship), or a reptile, amphibian, or bird (e.g., a snake, a frog, a chicken, a turkey, or other reptile, amphibian, or bird raised for human consumption or companionship).
The invention provides several advantages. For example, the methods can define for pharmaceutical companies and physicians a drug concentration (i.e., MPC), which, if maintained in patient serum during treatment, will severely restrict, if not eliminate, the selection of resistant mutants (FIG. 2). Moreover, the MSW, which varies among pathogen species and antibiotics, can help guide suppliers, clinicians and/or researchers to the drug least likely to select resistant mutants through calculation of the window index (FIG. 2). The methods can additionally guide the development of new drugs, as the methods enable rapid identification of drugs that have relatively small window indices, affording a reduction in the likelihood that drug-resistant pathogen strains will arise and proliferate. Since MPC, MIC, and WI can be measured without knowledge of the mechanism of drug action and without having in hand resistant mutants to test, the WI and MPC concepts can be used at very early stages of drug development to find drugs that are least likely to allow resistance. WI and MPC can thus be used for the identification of superior compounds for drug development. MPC and WI data can also provide quantitative guidelines for clinicians, researchers, and pharmaceutical companies. Clinicians and researchers can use the data for selection and dosing of drugs. For example, tp (FIG. 1) is useful for dosing since it indicates how often a drug must be taken to keep serum concentrations above MPC. Researchers and pharmaceutical companies can use the data to guide development of drugs and dosing proposals and regimens.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although suitable methods and materials are described below, other methods and materials, similar or equivalent to those described herein, can also be used in the practice or testing of the present invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.